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Drug discovery in the postgenomic era will lead researchers to focus on the proteins and biological mechanisms that cause disease as a way to develop new types of treatments that are safer and more effective
BY DENISE MYSHKO
When the initial map of the human genome was first published in February 2001, it quickly became evident that humans are both more and less complicated than researchers first thought. One of the surprises to come from the draft of the DNA sequence — which was published by scientists from the Human Genome Prod uct, sponsored by the U.S. Department of Energy, and Celera Genomics — was that there are only about 30,000 to 40,000 or so genes, instead of the 80,000 to 140,000 that had been predicted. This is only twice as many as those of a tiny transparent worm. Although the numbers of genes were fewer than scientists anticipated, they trigger far more complex biological processes than anyone imagined. These complex processes are created by the interactions of pro teins, which are produced by genes. Proteins are large, complex molecules made up of smaller subunits called amino acids. It is the proteins that create the millions of chemical reactions in the body, regulating the activities of cells, tissues, and organs. It is estimated that there are as many as 100,000 proteins and a million or more protein variations in the human body. Humans have on average three times as many kinds of proteins as the fly or worm because of mRNA transcript “alternative splicing” and chemical modifications to the proteins. This process can yield differ ent protein products from the same gene. Source: ornl.gov/hgmis 27 PharmaVOICE Ap r i l 2 002 BEYOND genomics While sequencing the genome was a sig nificant milestone and has provided researchers with much insight, it does not tell us the function of proteins or how proteins interact. The next logical step — and the next challenge — is to use the information gleaned from genomics research to study and map the proteins that are produced by genes and to determine their specific function. But mapping the human proteome — that is, the constellation of proteins in a cell — is likely to be an even more complicated process given the vast number of proteins and their interactions with each other. And unlike the relatively unchanging genome, the dynamic proteome changes from moment to moment in response to tens of thousands of intracellu lar and extracellular environmental signals. Enter proteomics, the study of protein expression and proteintoprotein interactions. Understanding how proteins function is the key to understanding how cells become dis eased. And understanding how proteins inter act with one another in biochemical networks in disease will provide valuable insight into developing new therapeutic modalities. “It’s always been clear to everybody in pro tein research that genomics was necessary but not sufficient,” says William E. Rich, Ph.D., president and CEO of Ciphergen Biosystems Inc. “Of course, investors have been a little dis appointed because they thought that once we knew the whole genome, drugs would just start flowing out the back end. That hasn’t happened and it won’t happen because proteins are what implement the disease and are the targets of the drugs to eliminate disease. So, if we don’t under stand the proteome, we won’t be able to make new drugs. The real work has just begun.” Proteomics will provide new insight into, and understanding of, complex cellular pro cesses. In fact, the inability to identify drug tar gets by examining a gene sequence has created a gap between genomics and drug discovery, say researchers at Kalorama Information. The true value of genomics will be realized only after protein function has been determined. In fact, after more than a decade of research, gene therapy remains highly experimental. While genetherapy research aims to correct a mal functioning gene so that the cell can function properly, protein research is likely to have broader implications by expanding the poten tial for new drug targets. “Proteomics will give us a much more detailed understanding of the molecular basis of disease,” says Norrie Russell, Ph.D., president and CEO of Lynx Therapeutics Inc., which develops technologies for gene expression. “Because many of these proteins will be only slightly different from one another, understand ing these fine differences will help us under stand the complexity of the molecular basis of disease. Armed with this understanding of the molecular basis of disease, the pharmaceutical industry will be able to design better drugs.” The promise for future research is to use data about genes and their proteins to design and develop small molecule and antibody products that target only the specific proteins involved in the disease of interest. This will lead to more effective treatments with fewer side effects. While most small molecule drugs now aim to interact with proteins as a way to change a func tion of a cell, they often are not specific enough to treat only the diseased cells. “Without a doubt, proteomics will change drug discovery, creating more and better qual ity targets that are validated, allowing chemi cals or proteins to interact with them for greater effect,” says Bill Kridel managing director of Ferghana Partners, an investment banking company. Proteomics is likely to have a bigger impact on the biotechnology industry, leading to the development of antibody products, says Dr. Samie Jaffrey, M.D., Ph.D., professor in the department of pharmacology, Cornell Univer sity’s Weill Medical College in New York. It may be difficult for pharmaceutical companies to develop small molecule drugs based on pro teomics research, he says. “Knowing the pro tein target doesn’t necessarily mean that com panies will be able to come up with a small molecule drug. Developing a small molecule drug is the hardest thing in the pharmaceuti cal industry. This is the part that takes years.” One bottleneck, he says, is in testing poten tial small molecules against targets. “There E Without a doubt, proteomics will change drug discovery, creating more and better targets that are validated, allowing chemicals or proteins to interact with them for greater effect. WILLIAM KRIDEL T 28 Ap r i l 2002 PharmaVOICE BEYOND genomics haven’t been any public methods that have been described to come up with drugs using pro teomics research. Proteomics in either of its two flavors — looking at expression profiles of pro teins or functional proteomics (which looks at how the properties of proteins change) — ulti mately give information about proteins, which often are the subject of the biotech industry.” Nevertheless, Dr. Jaffrey says there is a role for proteomics in helping the pharmaceutical industry identify potential targets. Proteomics research also is expected to add value to drug discovery by cutting total research and development costs. Proteomics has the potential to rapidly reveal novel drug targets and could be used during preclinical research to monitor cellular effects of lead compounds, says Eric Gay, an industry analyst with Frost & Sullivan. By finding new drug targets faster, researchers will be able to cut the associated costs of drug discovery. By “failing” faster — that is by determining which drug candidates not to pursue — companies could save about $100 million per drug on average if they could determine just one out of 10 cases against pur suing a target in the first place, according to a report by the Boston Consulting Group. In addition, proteomics promises to increase the number of potential drug targets 10fold, which expands the possible number of compounds that could be designed, accord ing to researchers at Frost & Sullivan. “There was so much attention paid to the sequencing of the human genome, but the true point of intervention inside the cell is going to be at the level of the protein,” Mr. Gay says. “So proteomics will give a much more direct indi cation in designing a drug.” Advances in genomics and proteomics also may provide an opportunity to segment patient populations more distinctly, leading to preven tive medicines and associated prolonged treat ment of patients with longerterm conditions. Dr. Paul L. Herrling, head of global research at Novartis Pharma AG, says this a positive for the industry. “Genomics and new biology will lead to new `blockbuster’ opportunities,” he says. “The clinical insights gained as we go through the process of rational drug design for one therapeutic area provide a better under standing of common biological mechanisms underlying different diseases. Thus, many of the new drugs will likely be able to treat multiple diseases simultaneously. We also believe that as these targets are identified, we should be able to develop more preventive therapies, so that treatment could start before symptoms appear.” The Proteomics Market Advancing technologies allow for a faster and more accurate study of proteins and their functions. Proteomics aims to produce high resolution protein maps for a given organism. It enables scientists to analyze changes in pro teins and compare normal and diseased tissues and cells. This research, however, requires sophisticated instruments and information systems — such as separation devices, robotics, mass spectrometers, and other tech niques for identifying and characterizing pro teins and simulating the behavior of disease. “Proteomics has been undergoing a change in the last few years,” says Gregg Morin, Ph.D., VP of biology at MDS Proteomics. “It is evolving from twodimensional gels and looking at whether a protein is expressed in one tissue versus another to an area of higher sensi tivity, looking at protein interactions and employing more mass spectrometry.” The leading technologies in proteomics are twodimensional gel electrophoresis and mass spectrometry, which separate and characterize proteins; protein chips, which offer a simple way to measure many proteins in common samples; and sophisticated bioinformatics tools to handle the data. The market for these tech nologies is expected to reach $2 billion this year and soar to $6 billion in five years, accord ing to Kalorama researchers. These technolo gies include protein chips, proteintoprotein interaction maps, protein databases, and bio logical assays. Analysts say most large pharmaceutical companies have in some way invested in pro teomics research either inter nally or through alliances with one or several of the PROTEOMICS TECHNOLOGIES PROMISE THEDISCOVERY ANDDEVELOPMENTOF THERAPEUTICS TARGETED TO THE SPE CIFIC PROTEINS IMPLICATED INDISEASE. While many of the bestselling products affect proteins,they often are not specific to the particular proteins involved in disease. The hope is that functional genomics and proteomics efforts can lead to more prod ucts that have fewer side effects and are more effective. One of the first small molecule drugs to target only the specific proteins involved in disease was developed by Novartis Pharma. Gleevec (imatinib) was approved in the U.S. in May 2001 to treat chronic myeloid leukemia (CML) and in February 2002 to treat malignant gastrointestinal stromal tumors.The product is known as Glivec out side the U.S. Although Gleevec was not dis covered using current proteomics technolo gy, the hope is that proteomics can lead to the discovery of other small molecule drugs. In the early 1980s, Novartis (then Ciba Geigy) started a cancer research program to investigate medicines that might selective ly inhibit specific kinases. Kinases regulate cell growth and differentiation, and they have been found to play a part in the devel opment of many types of cancer.This was a departure from cancer research efforts at the time, which was mostly aimed to pre vent the growth of cancerous cells, but because of an insufficient specificity also destroyed healthy dividing cells needed to sustain life thereby limiting their use and resulting in severe side effects. This research — headed by Dr. Alex Matter — led to the discovery in early 1990 of the first BcrAbl inhibitors, the pro tein kinases that play a role in chronic myeloid leukemia. In CML, an abnormal chromo some (called the Philadelphia chromosome) produces the protein BcrAbl, a tyrosine kinase. In the case of leukemia, BcrAbl changes a genetic signal to overproduce white blood cells. Then the team set to work on improving compounds that might specifically inhibit BcrAbl kinase activity. After two years, the team discovered Gleevec. Clinical trials showed that 88% of patients had their white blood cell counts return to normaland49%had either a disappearance or significant reduction of the Philadelphia chromosome. Gleevec was developed using rational drug design,says Dr.Paul L.Herrling,worldwide head of discovery, Novartis Pharma AG. “The original observation of this chromosome translocation in this particular leukemia was known since the 1960s.The reason it was known since the 1960s is that it could be seen through a microscope. We did not need sophisticated biology to see the chromosomes of these cells. But it took 30 years to understand the meaning of this translocation. This was done in the early 1990s Rational Drug Design in Action 29 PharmaVOICE Ap r i l 2 002 P BEYOND genomics many companies developing software and pro teomics instruments. Novartis, for example, is establishing new units, including the Drug Discovery Centre, focused on specific gene and protein families to search for drug targets. The integration of the Novartis Functional Genomics and Disease network is intended to lead to new kinds of treatments. The proteomics technology market is dynamic and fast growing. Kalorama Informa tion has identified at least 50 companies devel oping such technologies. Even large technology companies such as IBM and Oracle are working to create new technologies for proteomics research. IBM has several agreements in this area. The company, through an effort called Blue Gene, has committed $100 million over five years to computational biology. Among IBM’s alliances is one with Pro teome Systems Ltd. in Sydney, Australia, to advance protein research. IBM is providing the information technology backbone for Pro teome’s systems, including ProteomIQ. The new platform offers a comprehensive suite of software, instruments, and technologies for integrating, analyzing, and managing a full range of protein data. IBM and MDS Proteomics also have an agreement to establish a public research database. This alliance leverages IBM’s compu tational biology expertise and MDS’s capabili ties in protein identification and analysis. In other notable deals involving large tech nology companies, Caprion Pharmaceuticals Inc., San Diego, formed an alliance with Sun Microsystems, Oracle Veritas, and CGI to develop a data warehouse for Caprion’s high speed protein analysis system. Bioinformatics will be especially challenging. The large volume of available genomic and pro teomic data is increasing exponentially, and the need for protein databases is obvious. One initia tive is the Human Proteomics Initiative (HPI), which was launched two years ago by the Swiss Institute of Bioinformatics, a nonprofit academ ic institute, and the European Bioinformatics Institute. The ExPASy (Expert Protein Analysis System) is a server dedicated to the analysis of protein sequences and structures, including SWISSPROT, a protein knowledgebase. In the U.S., the North Carolina Supercomputing Cen ter (NCSC), a division of MCNC — a nonprofit corporation that offers costeffective access to advanced electronic and information technolo gies and services for businesses, state, and federal government agencies and North Carolina’s edu cation communities — was named in January as the U.S. mirror site for ExPASy. Another effort to develop a database of human proteins is being made by Confirmant Ltd. in Santa Clara, Calif., a joint venture formed last year between Marconi Plc. and Oxford GlycoSciences Plc. The company’s Pro tein Atlas of the Human Genome so far con tains information on 7,000 genes as well as the protein variants they encode. By June 2002, when the product’s commercial release is planned, it is expected to contain more than 10,000 proteinencoding genes. The data con tained in the Protein Atlas was derived from proprietary technologies developed by Oxford GlycoSciences in Oxford, United Kingdom. The market for bioinformatics technology is expected to experience increased growth and expansion. As the amount of protein informa tion increases, the need for data mining and knowledge discovery tools from the bioinfor matics arena will propel this market. It is pre dicted that the percentage of R&D budgets spent on bioinformatics for proteomics will increase from 0.2% to 1.5% by middecade, according to Frost & Sullivan. Proteomics technologies also are expected to contribute to the diagnostic market. In fact, the market for molecular diagnostics, includ ing genetic testing and analysis of protein biomarkers is expected to grow rapidly. Total genetic testing revenue is expected to reach $877 million by 2006, up from $319.9 mil lion in 2000, according to Frost & Sullivan. Proteomics Challenges The proteomics effort is still in its infancy, however. The current technology needed to separate and characterize proteins — two dimensional gel analysis followed by the iden tification of proteins through mass spectrome by David Baltimore’s team and others using transgenic mice and new techniques in molec ular biology.They expressed the human patho logical bits of chromosome in mice and showed that they became leukemic.He had the proof of causality. Then researchers found that this new pathological protein is a switch that turns on cell division without allowing down regulation. “At the moment this was known, our scien tists wereaccumulating experience in modulat ing kinases,” he says. “It occurred to them that this was our perfect target for this cancer. Because of its specificity to only the diseased cancerous cells, it did not affect healthy ones. We applied medicinal chemistry tools to find the right molecule to specifically inhibit the pathological protein (a kinase) and the result was Gleevec.” Gleevec, Dr. Herrling says, is designed to attack a modified pathological protein that exists only in the diseased cells. “Gleevec will only kill the cancer cells that have a particu lar chromosome translocation, the famous Philadelphia chromosome. The dramatic clinical benefits are a result of the tremen dous specificity.” Gleevec is the first of a kinase inhibitor that is relatively specific, says Dr. Samie Jaf frey,M.D.,Ph.D.,professor in the department of pharmacology at Cornell University’s Weill Medical College in New York.“Gleevec is wonderful because it has very few side effects because it doesn’t inhibit anything else. It just inhibits its major kinase target whereas other kinase inhibitors have failed in that regard because they inhibit multiple kinases and have multiple side effects. The reason is that all kinases look the same. So that any drug that binds to one kinase will almost always bind to another kinase, they are only incrementally different. And that incremental difference is being taking advantage of with Gleevec.” Gleevec is designed to attack a modified pathological protein that exists only in the diseased cells. DR.PAUL HERRLING 30 Ap r i l 2002 PharmaVOICE BEYOND genomics try — has limitations. Current methods are labor intensive, not sensitive enough, have a low throughput, and are not quantitative. “The proteome makes the genome look trivial,” Dr. Morin says. “There are about 35,000 to 40,000 genes in the genome. But that translates to somewhere on the order of 400,000 plus proteins in the cell, taking into account the differential processing of proteins. And proteins are designed to interact with each other and act on each other. All of that is com plicated by the fact that humans are multicel lular organisms with many different tissues. Each cell expresses a different range of proteins with a different set of interactions that all work in concert to control cell function.” “The complexity of proteomics is several orders of magnitude larger than genomics,” Dr. Herrling says. “Proteins are big and can fold in different ways. And they can combine in dif ferent ways to create different functions. This complexity of protein function is the reason why 30,000 genes can generate the complexi ty that is able to generate the millions of chem ical reactions needed to sustain life in the human body.” Better tools are needed to study protein measurement, protein structure, protein sequencing, as well as handling the huge amount of data that will be generated. These sophisticated tools are just emerging, especial ly either to replace or enhance twodimension al (2D) gel analysis for separating proteins. The 2D gel analysis is based on a 30yearold technology that uses a semisolid gel to stain and separate proteins based first on electric charge and then on size. “TwoD gel electrophoresis still has a blackmagic quality to it,” Dr. Russell says. “It takes a lot of experience and a lot of exper tise and a little bit of luck. The 2D gel elec trophoresis is not quantitative, so while a pro tein can be separated on the gel, it can’t be quantified accurately as to how much protein is in any given spot. It is not sensitive, because results only become apparent when the proce dure is finished. And low levels of protein do not get picked up.” In addition, he says, it is difficult to make these gels reproducible. “So if we do a disease prostate in one gel and normal prostate in another gel, it is very difficult to overlay them The human genome project’s historic achievement to sequence the 3 billion DNA bases that define the genetic mapofhumanbodyhasbeen completed.The humangenomepro ject provides a precise threegigabit mapof every gene that codes for the componentparts that define the human body. STATIC MODEL.However,the genome is a static molecule that merely contains the infor mation that directs another group of molecules’actions.The ultimate function of a gene is to provide the blueprint for the construction of proteins. Genomics — sequencing of DNA — is broadening the understanding of the genome and has defined the next step in bio logical research:determining the functions of proteins.Sequencing DNAwill tell the specif ic chemical composition of each protein but it will not be able to tell what function the pro tein actually performs or participates in when active.Thus, the focus of biological research in the postgenomic era is shifting to protein research. CATALOGING AND CHARACTERIZING. Proteomics attempts to catalog and charac terize proteins, compare variations in their expression levels under different conditions (notably sickness versus health), study their interactions, and identify their functional roles. Scientists believe there is a powerful distinction to be made between the molecular func tion of an isolated protein and the function of that protein in the complex cellular environ ment.Thus,one does not study each protein one at a time,as researchers have been doing for years, but rather the dynamic whole.Devising ways to accomplish this feat is one of the many challenges of proteomics. INTERFACING.Proteomics interfaces with, and complements,genomics to provide infor mation on quantitative protein expression in biological systems.This information will pro vide new insights into complex cellular processes and improve our understanding of cellu lar responses to external stimuli.Proteomics also promises to yield information on the ways in which cells respond to disease processes, leading to an understanding of disease at the molecular level and providing new opportunities for developing diagnostics and thera peutics. Moreover, scientists believe that proteomics also can be used to validate new ther apeutic agents by providing information about their effects on protein expression, thereby accelerating the process of drug discovery. SCOPE AND METHODOLOGY. Although proteomics is being promoted as a separate industry,it is in fact asetof technologies,whicharebeing increasingly used in combinationwith genomic technologies in the postgenomic era.Companies competing in the proteomics sec tor offer platform technologies for discovering or screening drug candidates or diagnostic markers.One of the ways emerging proteomics companies fund their enterprises is by selling their services to the pharmaceutical industry through an array of alliances and collaborations. Source: Kalorama Information, Kalorama’s Proteomics 50: Competing Technologies and Alliances in an Emerging Industry Executive Summary An Executive Summary The proteome makes the genome look trivial. There are about 35,000 to 40,000 genes in the genome. But that translates to somewhere in the order of 400,000 plus proteins in the cell, taking into account the differential processing of proteins. DR.GREGG MORIN BEYOND genomics 34 Ap r i l 2002 PharmaVOICE glass plate,” Dr. Russell says. “We do a first dimension separation in terms of size and the seconddimension separation in terms of charge. So we flip it around. The fluorescence is quantitative — the amount of fluorescence is in direct proportion to the amount of the protein.” Another emerging area is in the develop ment of protein biochips, or functional protein microarrays, to identify and characterize pro teins, as well as for profiling protein expres sion. Like DNA chips, the goal is to analyze thousands of samples simultaneously. But pro teins are more difficult to attach to the chips. Ciphergen’s ProteinChip has been available since 1999. It comprises a ProteinChip Read er integrated with ProteinChip software and a PC to analyze proteins captured on the com pany’s protein microarrays. It incorporates the company’s proprietary SurfaceEnhanced Laser Desorption/Ionization, or SELDI, technology on a disposable chip with multiple protein capture sites. This system captures, separates, and quantitatively analyzes proteins directly from crude biological materials with minimal sample preparation, allowing researchers to compare proteins in the disease versus normal state and analyze protein interactions. The company also announced that it is working on a new generation prototype of ProteinChip that is expected to be used for smallmolecule screening. Dr. Rich says the ProteinChip is not a replacement for 2D gel analysis. “It is a com plementary approach to 2D gels because it sees part of the proteome that 2D gels miss. We do two main applications: we have expres sion profiling, where we look at differential expression proteins, and protein interactions, which are the main parts of proteomics.” Another company working on a protein chip is Haywood, Calif.based Zyomyx Inc. In January, the company announced that it had received a patent covering a fundamental pro tein chip technology for creating surfaces suit ed to the study of protein interactions. The Protein Profiling Biochip system is designed to conduct simultaneous measurement of many proteins from very small samples of complex biological mixtures. The system under development will consist of a biochip designed to detect specific proteins, fluores cent reader, and assay components. Meanwhile, Bioarray Solutions LLC in Pis cataway, N.J., has developed a technology for the rapid analysis of DNA, proteins, and cells called LEAPS — lightcontrolled electroki netic assembly of particles near surfaces. This technology enables computercontrolled assembly of microparticles, or beads, and cells into planar arrays within a miniaturized enclosed fluid compartment on the surface of a semiconductor wafer. # PharmaVoice welcomes comments about this article. Email us at email@example.com. ERIC GAY. Industry analyst, Frost & Sulli van,San Antonio,Texas; Frost & Sullivan, with headquarters in NewYork, is a consulting company focused on emerging hightechnology and industrial markets DR.PAUL HERRLING. Head of global research, Novartis Pharma AG,Basel, Switzerland; Novartis is a world leader in healthcare DR.SAMIE JAFFREY,PH.D. Professor in the department of pharmacology,Cornell Weill Medical College, NewYork;Weill Medical College of Cornell University is a clinical and medical research center WILLIAM J. KRIDEL,JR.Managing direc tor, Ferghana Partners Ltd. and managing director, Ferghana Partners Inc.; Ferghana Partners is a lifesciences banking house with offices in London and NewYork Experts on this topic GREGGMORIN,PH.D.VP,biology,MDS Proteomics Inc.,Toronto, Ontario; MDS is a proteomicsbased drug discovery compa ny, whose goal is to help its partners increase productivity through rapidly iden tifying, triaging, and selecting more func tionally annotated drug targets in disease WILLIAM E. RICH,PH.D. President and CEO,Ciphergen Biosystems Inc., Fremont, Calif.; Ciphergen’s mission is to improve the understanding of disease processes through the use of advanced protein tech nology products and services to enable rapid discovery, characterization, and assays NORRIE RUSSELL,PH.D.President and CEO,Lynx Therapeutics Inc., Hayward,Calif; Lynx Therapeutics develops novel tech nologies for the discovery of gene expres sion patterns and genomic variations The ProteinChip is not a replacement for 2D gel analysis. It is a complementary approach to 2D gels because it sees part of the proteome that 2D gels miss. DR.WILLIAM RICH There is a role for proteomics in helping the pharmaceutical industry identify potential targets. DR.SAMIE JAFFREY